Cassettes for preserving natural and biological tissues

Abstract

Described herein are cassettes and methods of using these cassettes for cryopreserving biological materials (e.g., bioengineered constructs or native tissue samples) by vitrification while reducing or preventing the loss of viability associated with traditional preservation methods.

Description

[Technical Field]

[0001] [Government support] This invention was supported in whole or in part by grant 1R43TR003258-01 from the National Institutes of Health. The government has certain rights to this invention.

[0002] [Cross-reference with related applications] This application claims priority to U.S. Provisional Application No. 63 / 279,240, filed on November 15, 2021. The disclosures of the prior application are incorporated herein by reference in their entirety.

[0003] This disclosure relates to a method for cryopreserving natural tissues and bioengineered tissues, such as bioengineered constructs, while reducing or preventing the loss of viability associated with conventional preservation methods. The disclosure further relates to a specially designed cassette and a high-capacity system capable of preserving multiple natural tissues and bioengineered tissues, such as bioengineered constructs, at once {for example, by using a specially designed cassette capable of holding a predetermined number (e.g., 6 to 384, e.g., 24) well inserts containing natural tissues and bioengineered tissues, such as bioengineered constructs, at once}. [Background technology]

[0004] Over the past several decades, preservation methods and techniques have been developed for saving eukaryotic tissues and cells. These methods and techniques are aimed at preserving various eukaryotic cells for a certain period of time in engineered extracellular matrices (substrates), engineered tissues, and native tissues in a way that allows these preserved tissues to be used at a later date, such as for implantation or transplantation into patients, or for screening bioassays of drugs or chemicals.

[0005] While such preservation methods and techniques are widely applicable in both basic and translational research, maintaining the properties of biomaterials during preservation (e.g., cell viability and extracellular matrix integrity) remains a challenge, especially for biotechnological components. For example, current techniques have been observed to significantly reduce extracellular matrix permeability and tissue cell viability, which can lead to inefficient function of biomaterials after retrieval from storage.

[0006] The development of in vitro assays for various human tissues can address the need to reduce the number of animals used in research, find more cost-effective methods for screening new drugs, compounds, or techniques, and discover in vitro assays that more predict in vivo responses in humans. Increasing research supports the use of tissue-engineered constructs, made from various tissues beyond skin, in toxicity testing. In this regard, 3D human tissue models and tissue-equivalent constructs are replacing animal models in toxicity prediction screening and drug discovery models. These models are lower cost and undoubtedly more closely resemble natural human responses than comparable animal models.

[0007] For example, companies involved in the manufacture of cosmetics, chemicals, household goods, and pharmaceuticals are beginning to use tissue-equivalent components as an alternative to animal testing (Non-patent Literature 1: Afaq et al., Protective effect of pomegranate-derived products against UVB-mediated damage in human reconstructed skin, Exp Dermatol., 18(6):553-61 (2009); Non-patent Literature 2: Felippi et al., Safety and efficacy of antioxidant-containing nanoparticles for anti-aging applications, J Biomed Nanotechnol, 8(2):316-321 (2012); Non-patent Literature 3: Jirova et al., Comparison of human skin irritation patch test data with in vitro skin irritation assay and animal data, Contact Dermatitis, 62(2):109-16 (2010); Non-patent Literature 4: Kaluzhny et al., Development of EpiOcular® eye irritation test for hazard identification and labeling of eye irritant chemicals in accordance with the requirements of the EU Cosmetics Directive and REACH Act, Altern Lab Anim, 39(4):339-64 (2011); Non-Patent Literature 5: Kolle et al., In-house verification of a combination of the EpiOcular® eye irritation test and the bovine corneal opacity and permeability test for eye irritation evaluation, Altern Lab Anim, 39(4):365-87 (2011); Non-Patent Literature 6: Ren et al., Use of EpiAirway for characterizing long-term host-pathogen interactions, J Vis Exp. 55: e3261 (2011); Non-Patent Literature 7: Scheel et al., Classification and labeling of industrial products with extreme pH using in vitro methods for evaluating skin and eye irritation and corrosiveness using an evidence-weighted approach, Toxicol In Vitro, 25(7):1435-47 (2011); Non-Patent Literature 8: Sharma et al., Efficacy of echinacea in a three-dimensional tissue model of human airway epithelium, Phytother Res., 24(6):900-4(2010)). The entirety of the disclosures in these publications is incorporated into this application, for example, to more completely describe the state of the art to which this disclosure relates.

[0008] Since 2009, European Union (EU) regulations prohibiting the use of animals for collecting toxicological data on cosmetic ingredients have validated MatTek's epidermal-like in vitro tissue models for use in toxicity testing of cosmetic ingredients. With the ban on animal toxicity testing expected to soon follow for other types of compounds, such as pharmaceuticals, the demand for in vitro models of all tissue types is increasing. Tissue components are generally made from a single cell type but contain multiple cell layers. In some cases, components with multiple cell types have also been developed. Therefore, while more complex than monolayer cells in a dish or plate, they are entirely different from actual tissues such as venous segments or cartilage fragments.

[0009] Such biotechnically engineered human tissues are currently manufactured in custom batches by industrial suppliers and must be used immediately upon receipt. This means that biotechnically engineered components are generally made to order, requiring a lead time of several weeks before shipment. Shipments are carried overnight at 4°C, and biotechnically engineered tissues, such as biotechnically engineered components, must be used within a finite period (1-2 weeks) to obtain best results. Due to the short shelf life of such cell materials, quality control testing may not be completed before shipment and may have to be performed retrospectively. If the results of such quality control testing are delayed, customers may use equivalent tissues in their research projects, expending significant resources, time, and effort, only to discover that the batch ultimately does not meet the prescribed quality control standards. Availability can also be an issue if effective components become unavailable for various reasons, such as production problems due to weather or a shortage of suitable starting materials. In such cases, without this tool for toxicity testing, the development of pharmaceuticals and other compounds would be put on hold, resulting in wasted time and money due to inactivity. Therefore, improved methodologies are needed for preserving biotechnological structures, such as biotechnological components, for future use.

[0010] Currently, cryopreservation is not used to preserve these tissues for later use. Cryopreservation would dramatically increase the availability of biotechnological tissues such as biotechnological components, expand the market, reduce overall manufacturing costs through economies of scale, and consequently lead to more efficient shipments and delivery to customers.

[0011] In other words, the cryopreservation method for biotechnological tissues, such as biotechnological components, eliminates the lead time required to manufacture biotechnological tissues on demand, enables quality control checks of inventory before shipment, and reduces costs through economies of scale. End users can schedule experiments more flexibly without worrying about the availability or quality of biotechnological tissues. Customers can also order large quantities of the same batch of products to better control uniformity within their research. Validated cryopreserved tissue components can be shipped to any laboratory in the food, pharmaceutical, cosmetic, or chemical industries and can be conveniently used as an alternative to in vivo testing.

[0012] While preserving biotechnological tissues, such as biotechnological components, may seem straightforward, conventional cryopreservation methods fail to produce viable biotechnological tissues (generally <50%).

[0013] The effectiveness of vitrification strategies has been repeatedly demonstrated for the preservation of structure, extracellular matrix, and viability of both natural and biotechnological tissues, from blood vessels and heart valves to encapsulated cells and cartilage (Non-Patent Literature 9: Brockbank et al., Quantitative Analysis of Vitrified Autologous Venous Artery Bypass Grafts, Cell Preservation Techniques, 5 (2) (2007); 68-76; Non-Patent Literature 10: Brockbank et al., Vitrification of Porcine Articular Cartilage, Cryobiology 60, 217-221, http: / / www.pubmedcentral.gov / articlerender.fcgi?artid=2834839 (2010); Non-Patent Literature 11: Dahl et al., Potential of Vitrification as a Method for Preserving Tissue-Modified Blood Vessels, Tissue Eng., 12(2):291-300 (2006); Non-Patent Literature 12: Schenke-Layland et al., Optimal Preservation of Extracellular Matrix in Cardiac Tissue: Effects on Long-Term Durability of Grafts, Annals of Thoracic Surgery, 83:1641-1650 (2007); Non-patent Literature 13: Song et al., Vitreous cryopreservation to maintain the function of artificial blood vessels, Nature Biotechnology, 8(3):296-9, Epub 2000 / 03 / 04, doi:10.1038 / 73737, PubMed PMID: 10700144 (2000); Non-patent Literature 14: Song et al., Vitrification of tissue-engineered pancreatic substitutes, Transplantation Proceedings, 37(1):253-255 (2005); and Non-patent Literature 15: Song et al., Vitreous preservation of rabbit articular cartilage, Cell Preservation Technology, 2(1); 67-74 (2004). The entirety of the disclosures in these publications are incorporated into this application by reference, for example, to more fully illustrate the technical state to which this disclosure relates. Vitrification is the solidification of a liquid without crystallization. As cooling progresses, the molecular motion of the liquid permeating the tissue decreases. Ultimately, a "stationary liquid" state known as glass is achieved. This conversion from liquid to glass is called vitrification (derived from the Greek word "vitri," meaning glass).Vitrification can be achieved by adjusting the solute composition and cooling rate to prevent ice crystal nucleation and growth. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Afaq et al., Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin, Exp Dermatol., 18(6): 553-61 (2009) [Non-Patent Document 2] Felippi et al., Safety and efficacy of antioxidants-loaded nanoparticles for an anti-aging application, J Biomed Nanotechnol, 8(2): 316- 321 (2012) [Non-Patent Document 3] Jirova et al., Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data, Contact Dermatitis, 62(2): 109-16 (2010) [Non-Patent Document 4] Kaluzhny et al., Development of the EpiOcular(TM) eye irritation test for hazard identification and labeling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation, Altern Lab Anim, 39(4): 339-64 (2011) [Non-Patent Document 5] Kolle et al., In-house validation of the EpiOcular(TM) eye irritation test and its combination with the bovine corneal opacity and permeability test for the assessment of ocular irritation, Altern Lab Anim, 39(4): 365-87 (2011) [Non-Patent Document 6] Ren et al., Use of the EpiAirway model for characterizing long-term host-pathogen interactions. J Vis Exp. 55: e3261 (2011) [Non-Patent Document 7] Scheel et al., Classification and labeling of industrial products with extreme pH by making use of in vitro methods for the assessment of skin and eye irritation and corrosion in a weight of evidence approach, Toxicol In Vitro, 25(7): 1435-47 (2011) [Non-Patent Document 8] Sharma et al., The efficacy of Ehcinacea in a 3-D tissue model of human airway epithelium, Phytother Res., 24(6): 900-4 (2010)

Non-patent Document 9

Non-patent Document 10

Non-patent Document 11

Non-patent Document 12

Non-patent Document 13

[0015] However, a drawback of using high concentrations of cryoprotective agents (CPAs), which are commonly used in the vitrification process, is that they can be cytotoxic. One advantage of vitrification, however, is that the biomaterials trapped within the vitreous matrix do not degrade over time, thus avoiding the effects of biological damage associated with freezing.

[0016] Thus, while the cryopreservation of individual components has been successful, there is a need to improve the yield of viable tissue components (i.e., significantly higher than 50%). Furthermore, although it is possible to preserve multiple components at once (previously only possible when two technicians worked together), there is a greater need for a high-capacity system that can preserve multiple components simultaneously and achieve a yield of viable tissue components significantly higher than 50%.

[0017] The inventors of this disclosure have developed a methodology that enables the achievement of this (i.e., preservation of individual natural or biotechnological tissues, such as biotechnological constructs, or multiple natural and / or biotechnically produced tissues, e.g., biotechnically produced constructs, where the yield of viable tissue constructs is significantly higher than 50%). In some embodiments, this involves using a cassette of this disclosure that can hold and successfully preserve a large number of well inserts (e.g., 24 or up to 384) at once, including natural and / or biotechnological constructs, such as biotechnological constructs. The cassette has two parts that interlock to hold the inserts. The top has a placeholder for holding the inserts in a configuration that conforms to the well arrangement of a well plate. This configuration facilitates the transfer of biomaterials (e.g., natural and / or biotechnological tissues, such as biotechnological constructs) from the cassette to the plate as needed. Holes are present in both parts of the cassette, allowing the solution (such as the solution used during the vitrification step) to flow easily into and around each insert and biomaterial (e.g., natural and / or biotechnical tissues such as biotechnical components). In some embodiments, the cassette can be immersed in the vitrification solution during the cryoprotectant injection / removal stage and the actual vitrification. The placeholder can also be easily modified to hold fragments of native tissue for preservation. The cassette can be easily adapted for automation of the entire vitrification process. [Means for solving the problem]

[0018] This specification describes methods for preserving biomaterials (e.g., natural and / or biotechnological tissues, such as biotechnological components) while reducing or preventing the loss of viability associated with conventional preservation methods. This specification also describes cassettes for cryopreserving biomaterials (e.g., natural and / or biotechnological tissues, such as biotechnological components) and methods for using these cassettes.

[0019] In other words, the present invention describes a multi-sample cassette comprising a first part and a second part configured to fit together via a releasable locking mechanism, wherein the second part comprises one or more inserts and a plurality of placeholders, each of the plurality of placeholders configured to receive one of the one or more inserts, each of the one or more inserts comprising at least one biotechnological component or natural tissue sample, wherein the first part and the second part comprise a plurality of openings configured to allow a solution to flow into and around each of the one or more inserts and the at least one biotechnological component or natural tissue sample, the configuration of the placeholders in the cassette being set to mimic the configuration of wells in a plate having a predetermined number of wells, the predetermined number of wells being in the range of 6 to 384. [Brief explanation of the drawing]

[0020] [Figure 1] A schematic diagram of a cassette according to an embodiment is shown. [Figure 2A] This is a schematic diagram showing the cassette as a whole according to the embodiment, illustrating how the biomaterial fits into the cassette. [Figure 2B] Figure 2A shows a side view of the cassette in the closed position. [Figure 2C] Figure 2B shows a side view of the cassette with the lid removed. [Figure 2D] Figure 2B shows a side view of the cassette, which has two sides, with the biological material sample positioned on one side. [Figure 2E] Figure 2D shows side views of both sides of the cassette. [Figure 3] This is a photograph of a cassette according to an embodiment. [Figure 4] This chart shows the survival rate of epidermal components after protocol modification according to the disclosed examples. [Figure 5] This chart shows the survival rates of fresh and vitrified epidermis after exposure to Triton-X100, according to the disclosed examples. [Figure 6] This chart illustrates the viability of several components after vitrification in vials and deep-well plates according to the disclosed examples. [Figure 7] This chart illustrates the survival rates of various components after storage, according to the disclosed embodiments. [Figure 8] This chart illustrates the survival rates in deep well plates and cassettes according to the disclosed examples. [Modes for carrying out the invention]

[0021] The disclosed cassettes and methods can be more readily understood by referring to the following detailed descriptions of specific embodiments, the examples included herein, and the figures and their descriptions.

[0022] In this specification and the subsequent claims, numerous terms are defined as having the following meanings:

[0023] As used herein, the term “room temperature” refers to a temperature of approximately 18°C ​​to approximately 25°C at standard pressure. In various examples, room temperature may be approximately 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C.

[0024] As used herein, the term “vitrification” refers to solidification without the formation of ice crystals, or without substantial ice crystal formation. In some embodiments, the sample to be preserved (e.g., tissue or cellular material) may be vitrified such that vitrification and / or vitreous cryopreservation (the entire process from initial cooling to completion of rewarming) may be achieved without ice crystal formation. In some embodiments, the sample to be preserved (e.g., tissue or cellular material) may be vitrified such that vitrification and / or vitreous cryopreservation may be achieved in which solidification of the sample to be preserved (e.g., tissue or cellular material) may occur without substantial ice crystal formation (i.e., vitrification and / or vitreous cryopreservation (the entire process from initial cooling to completion of rewarming) may be achieved in the presence of a small or limited amount of ice, less than the amount that would damage the tissue).

[0025] As used herein, the sample or biomaterial to be preserved (e.g., natural tissue or biotechnically treated tissue such as biotechnically treated components) has a glass transition temperature (T g When it reaches ), vitrification occurs. During the vitrification process, the viscosity of the cryoprotectant solution increases significantly as the temperature decreases, inhibiting ice nucleation and growth. Generally, the lowest temperature at which a solution can be supercooled without freezing is the homogeneous nucleation temperature T. h At this temperature, ice crystals nucleate and grow, and a crystalline solid is formed from the solution. The vitrification solution has a glass transition temperature T g At this temperature, the solute vitrifies, meaning it becomes an amorphous solid.

[0026] As used herein, “glass transition temperature” refers to the glass transition temperature of a solution or formulation under the conditions under which the process is carried out. Generally, the methodologies of this disclosure are carried out at physiological pressure. However, higher pressures may be used, provided that the sample being preserved (e.g., tissue or cell material) is not significantly damaged thereby.

[0027] As used herein, the term “cryoprotectant” means a chemical substance that minimizes ice crystal formation within and around tissues / organs when the tissue is cooled to below freezing point, resulting in substantially no damage to the tissue / organ after warming compared to the effect of cooling without a cryoprotectant.

[0028] As used herein, the term “biomaterial” includes natural and / or bioengineered cells or tissues, or bioengineered constructs of non-plant, mammalian eukaryotes. As used herein, the terms “tissue,” “tissues,” “construct,” or “constructs” consist of and / or are derived from any type of cell and any combination thereof, such as ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, connective tissue, cardiac tissue, muscle, cartilage, and bone tissue, endocrine tissue, skin, nerve tissue, somatic cells (including any type of cell in a tissue or organ), fibroblasts, keratinocytes, hepatocytes, chondrocytes, smooth muscle cells, stem cells, progenitor cells, oocytes, germ cells, etc.

[0029] The terms “tissue,” “tissues,” “construct,” or “constructs” may also include adipose tissue or dental pulp tissue. In some embodiments, “tissue” or “tissues” may be obtained from humans, such as human liver, human lung, human kidney, human intestine, human heart, human pancreas, human testis, human placenta, human thymus, human adrenal gland, human artery, human vein, human nerve, human skin, human lymph node, human bone, or human skeletal muscle. In some embodiments, “construct” or “constructs” may be obtained from and / or derived from human tissues or organs, such as human liver, human lung, human kidney, human intestine, human heart, human pancreas, human testis, human placenta, human thymus, human adrenal gland, human artery, human vein, human nerve, human skin, human lymph node, human bone, or human skeletal muscle.

[0030] As used herein, the term “functional after cryopreservation” in relation to cryopreserved biomaterials means that cryopreserved biomaterials, such as natural and / or biotechnically modified cells and tissues, e.g., biotechnically modified components, retain an acceptable and / or intended function after cryopreservation (e.g., that they can function as models for predictive toxicity screening and for drug discovery). In some embodiments, the cellular material of a cryopreserved biomaterial retains all of its intended function. In some embodiments, the cellular cryopreserved biomaterial preserved by the methods of the present disclosure retains at least 50% of the intended function, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., 100%, e.g., and so on. For example, it may be important to maintain / preserve the physiological function(s) of biomaterials (e.g., natural tissues and / or bioengineered tissues such as bioengineered components) that functions as a model for predictive toxicity screening and drug discovery, and / or as the ability of tissue (e.g., transplanted tissue) to integrate with surrounding tissue, in addition to maintaining cell viability.

[0031] Described herein are viable biomaterials (e.g., natural and / or biotechnically engineered tissues such as biotechnically engineered components) and methods for preserving / storing such components, such as within the cassettes of this disclosure.

[0032] In certain embodiments, these biomaterials include eukaryotic cells (which may be either engineered tissues or native tissues, or a combination of both), and the methods described herein include preserving / storing these biomaterials(s) (e.g., native tissues and / or biotechnical tissues, such as biotechnical components) in such a manner that the loss of biomaterial properties (e.g., tissue / cell viability, extracellular matrix integrity, or a combination thereof) that occurs either during storage or after removal of the biomaterials (e.g., native tissues and / or biotechnical tissues, such as biotechnical components) from storage is reduced or prevented. In embodiments, these biomaterials (e.g., native tissues and / or biotechnical tissues, such as biotechnical components) are placed in a solution or a series of solutions (e.g., to achieve a final cryoprotectant concentration), such as a pre-cooled (multiple) vitrification solution, which contains at least one agent, such as a cryoprotectant. Subsequently, the biomaterials (e.g., natural and / or biotechnological tissues, such as biotechnological components) placed in a solution containing at least one agent are stored within a specific temperature range until further use of these biomaterials is required. The concentration of at least one agent, such as a cryoprotectant, is optimized to maximize the biomaterial properties (e.g., cell viability and / or extracellular matrix integrity) of the biomaterials (e.g., natural and / or biotechnological tissues, such as biotechnological components).

[0033] When using the biomaterial(s) described herein (e.g., natural tissues and / or bioengineered tissues such as bioengineered components) in conjunction with the compositions and methods described herein, one objective is to prevent loss of cell viability and / or loss of extracellular matrix integrity.

[0034] For example, in certain embodiments, the object of this disclosure is to reduce or prevent the loss of cell viability in biomaterials. In certain embodiments, the compositions, cassettes, and methods described herein can be used to reduce or prevent various types of cell death, including but not limited to necrotic cell death, apoptotic cell death, autophagy (type II) cell death, annoikis, and necroptosis, and in certain embodiments, these types of cell death can be limited by the use of agents such as those further described below. Metabolic activity assays (e.g., the resazurin assay used to assess cell viability by measuring the oxidation / reduction reactions occurring within cells), various cell staining techniques (e.g., the trypan blue exclusion assay and vi / dead staining), immunohistochemistry, biochemistry, and various gene expression assays may be used to assess viability.

[0035] Furthermore, the integrity of the extracellular matrix can be determined based on permeability, water content, glycosaminoglycan content, or a combination thereof. In certain embodiments, one objective is to maintain at least one of permeability, water content, glycosaminoglycan content, or a combination thereof, while preserving the biomaterial(s) to prevent or reduce the loss of extracellular matrix integrity. When determining the integrity of the matrix of a biomaterial(s), a number of techniques known in the art can be used. These techniques include matrix conductivity assays for measuring permeability, water content, and glycosaminoglycan content, indentation tests, stress / strain tests, elasticity tests, RAMAN spectroscopy, and various microscopy techniques (such as laser scanning microscopy with second harmonic generation).

[0036] In some respects, when tissue matrices are used as biomaterials, preventing or reducing the loss of cell viability and the loss of extracellular matrix integrity is important for maintaining the structural integrity and normal biological function of the biomaterial.

[0037] For example, cartilage (either natural cartilage or bioengineered cartilage such as bioengineered cartilage components) contains chondrocytes (i.e., cells) and an extracellular matrix, the extracellular matrix consisting mainly of collagen fibers, proteoglycans, and elastin fibers. Chondrocyte viability and the integrity of the chondrocyte extracellular matrix are important for maintaining normal physiological biological functions in in vivo, ex vivo, and in vitro applications. For example, the extracellular matrix of cartilage provides structural integrity in vivo, maintains a certain level of rigidity, and functions in bone support, proper joint mobility, etc. In some aspects, the permeability of the extracellular matrix of cartilage is particularly important. For example, the permeability of cartilage plays a crucial role in maintaining the structural integrity of the extracellular matrix of cartilage and maintaining the viability of chondrocytes. In some embodiments, reduced permeability of the extracellular matrix of cartilage may be associated with increased chondrocyte viability and decreased structural integrity of the extracellular matrix of cartilage.

[0038] The biomaterials(s) described herein (e.g., natural tissues and / or biotechnological tissues such as biotechnological components) may be placed in a solution (e.g., a vitrification solution) designed to prevent or reduce the loss of properties of the biomaterials (e.g., cell viability, extracellular matrix integrity, or a combination thereof), and in some embodiments, this solution may be free of animal products (e.g., free of FBS) or may contain animal products (e.g., contain FBS). It should be noted that the following description and embodiments also apply to solutions containing animal products containing biomaterials. In some embodiments, the biomaterials(s) (e.g., natural tissues and / or biotechnological tissues such as biotechnological components) may be at least partially immersed in the solution (e.g., while the biomaterials(s) are contained in the cassette of this disclosure), and in other embodiments, the biomaterials may be fully immersed in the solution (e.g., while the biomaterials(s) are contained in the cassette of this disclosure).

[0039] In one embodiment, the solution may be an extracellular type solution containing at least one agent that prevents or reduces the loss of properties of the biomaterial (e.g., cell viability, extracellular matrix integrity, or a combination thereof). For example, an extracellular type solution may include an isotonic, plasma-like solution having ionic complement that mimics the normal extracellular environment of cells and tissues (e.g., natural tissues and / or bioengineered tissues such as bioengineered constructs) of the biomaterial (maybe multiple cells). These isotonic, plasma-like solutions may include cell culture media that provide various amino acids and metabolites to the biomaterial (e.g., cells and / or tissues) for nutritional support. Examples of cell culture media used in extracellular type solutions include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), αMEM, Glasgow MEM, Hum F10, Hum F-12, Leibowitz L-15, Iscove Modified DMEM, DMEM / Hum F-12, and derivatives thereof. Extracellular solutions can be made free of animal products so that the cell solution does not contain animal products before biomaterials (e.g., natural and / or bioengineered tissues such as bioengineered components) are added to the cell solution. For example, if cell culture media are used, the cell culture media should not contain fetal bovine serum (FBS) or any other animal-derived products.

[0040] In certain embodiments, solutions (e.g., vitrification solutions) include intracellular solutions. Intracellular solutions may include, but are not limited to, isotonic solutions formulated to limit the passive exchange of water and ions between cells in biomaterials (e.g., natural and / or biotechnical tissues such as biotechnical components) and the intracellular solution during storage. For example, intracellular solutions may contain impermeable anions such as lactobionates or glucons, which partially replace chloride ions in the extracellular space and provide osmotic support to balance the intracellular oncological pressure generated by cytoplasmic macromolecules and their associated counterions trapped within the cell. Examples of intracellular solutions include, but are not limited to, VIASPAN® (i.e., Belzer's Solution) and UNISOL® (e.g., SPS-1). Similar to the extracellular solutions described above, intracellular solutions may be free of animal products.

[0041] To further supplement the solution and promote the viability of the biomaterial, additional agents / components may be added to the solution (e.g., vitrification solution). For example, these additional agents / components may provide additional nutritional support to the biomaterial(s) (e.g., natural and / or biotechnological tissues such as biotechnological components), thereby reducing or preventing the loss of viability of the biomaterial(s) (e.g., natural and / or biotechnological tissues such as biotechnological components). Examples of these additional agents / components include, but are not limited to, nutritional cocktails containing non-animal (i.e., synthetic) essential amino acids, synthetic non-essential amino acids, synthetic vitamins, synthetic lipids, synthetic carbohydrates, or any combination thereof. Examples of carbohydrates in a nutritional cocktail include saccharides and / or their derivatives (e.g., glucose, glycerol, sucrose, trehalose, fructose, galactose, maltose, lactose, etc.), or further combinations thereof. Examples of amino acids provided in the cocktail include, but are not limited to, glycine, L-arginine, L-cystine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, or any combination thereof. Examples of vitamins provided in the cocktail include, but are not limited to, choline, D-calcium, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, inositol, or any combination thereof. In some embodiments, the drug may contain one or more of Q-VD-OPH (quinoline-Val-Asp-difluorophenoxymethyl ketone), α-tocopherol, ferulic acid, curcumin, allene oxide synthase, and SDF-1.

[0042] In certain embodiments, the agent can reduce, for example, the loss of properties of biomaterials (e.g., cell viability and / or extracellular matrix integrity) by, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 99% or more compared to a control. In other words, the agent can substantially or completely suppress, for example, the loss of properties of biomaterials (e.g., natural tissues and / or biotechnically engineered tissues such as biotechnically engineered components) by, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% compared to a control.

[0043] In this particular embodiment, the solution is available in concentrations of 1 pM to 2 mM, 10 pM to 1 mM, 1 nM to 1 mM, 100 nM to 0.5 mM, 100 nM to 0.25 mM, 1 μM to 1 mM, 250 μM to 1 mM, 1 pM to 1000 μM, 1 pM to 500 μM, 1 pM to 30 μM, 1 pM to 1000 nM, 1 pM to 500 nM, 1 pM to 250 nM, 100 pM to 750 μM, 100 pM to 500 μM, 100 pM to 20 μM, 100 pM to 1000 nM, 1 pM to 750 nM, 1 pM to 500 nM, 1 pM to 250 nM, and 1 pM~1nM, 500pM~500μM, 500pM~250μM, 500pM~100μM, 500pM~10μM, 500pM~1000nM, 500pM~750nM, 500pM~500nM, 500pM~250nM, 500pM~10 0nM, 500pM~1nM, 1nM~1000μM, 1nM~750μM, 1nM~500μM, 1nM~250μM, 1nM~100μM, 1pM~1μM, 100nM~1000μM, 100nM~750μM, 100nM~500μM, 1 00nM~250μM, 100nM~100μM, 100pM~1μM, 250nM~1000μM, 250nM~750μM, 250nM~500μM, 250nM~250μM, 250nM~100μM, 250nM~1μM, 500nM~1 000μM, 500nM~750μM, 500nM~500μM, 500nM~250μM, 100nM~100μM, 500nM~1μM, 750nM~1000μM, 750nM~750μM, 750nM~500μM, 750nM~250μM M, 750nM~100μM, 750nM~1μM, 0.5μM~1000μM, 10μM~950μM, 20μM~900μM, 30μM~850μM, 40μM~800μM, 50μM~750μM, 60μM~700μM, 70μM~650 μM, 80 μM ~ 600 μM, 90 μM ~ 550 μM, 100 μM ~ 500 μM, 110 μM ~ 450 μM, 120 μM ~ 400 μM, 130 μM ~ 350 μM, 140 μM ~ 300 μM, 150 μM ~ 250 μM, 160 μM ~ 200 μM, 0.Concentrations (combined or individually) within the ranges of 5 μM to 100 μM, 1 μM to 90 μM, 5 μM to 90 μM, 10 μM to 85 μM, 10 μM to 75 μM, 20 μM to 85 μM, 20 μM to 65 μM, 30 μM to 70 μM, 30 μM to 50 μM, 40 μM to 80 μM, or 40 μM to 50 μM, including one or more such drugs, where any concentration occurring within the above ranges can also serve as the endpoint of the range.

[0044] Vitrification can be achieved using various cryoprotectant mixtures and cooling / warming conditions. Key variables should be optimized for each specific extracellular tissue matrix type of biomaterial (e.g., natural and / or bioengineered tissues, such as bioengineered components) and the size of individual biomaterials. The selection of cryoprotectant mixtures and the equilibration steps required for the addition and removal of cryoprotectants without excessive osmotic shock should be optimized based on the measured kinetics of cryoprotectant penetration in biomaterial samples, or by demonstrating viability and / or function. Freeze-replacement can also be employed to confirm that ice-free preservation has been achieved using a given protocol.

[0045] Embodiments may include a stepwise cooling process, for example, cooling a biomaterial(s) (e.g., bioengineered tissues such as natural tissues and / or bioengineered components) in a first solution containing a cryoprotectant to a first temperature (0 to +4°C) to -20°C, then further cooling to a second temperature in a second solution containing a cryoprotectant (at a higher concentration than the previous solution) at a temperature between the temperature of the first solution and -20°C, and this process may be repeated a third, fourth, fifth, sixth, seventh, and so on, until a desired cryoprotectant concentration and temperature are reached. In this regard, as will be discussed in more detail below, holes are present in both parts of the cassette of the present disclosure, allowing each solution to flow easily into and around each insert and biomaterial (and replace / exchange the previous solution). In some embodiments, the flow of each solution can be paused for a predetermined time so that the cassette can be immersed in each solution during the CPA injection / removal process and / or during the actual vitrification or storage.

[0046] The final cryoprotectant concentration of the vitrification solution may be achieved by immersing a biomaterial(s) (e.g., bioengineered tissue such as natural tissue and / or bioengineered constructs) in a first solution containing a first cryoprotectant concentration (e.g., while the biomaterial(s) are contained within the cassette of the disclosure), and then immersing the biomaterial(s) (e.g., while the biomaterials are contained within the cassette of the disclosure) in a second solution containing a second cryoprotectant concentration (which is higher than the first cryoprotectant concentration), and repeating this process for a third, fourth, fifth, sixth, seventh, and so on, until the desired concentration is achieved.

[0047] The solution / vitrification preparation may contain any combination of cryoprotectants. Suitable cryoprotectants include, in addition to those listed below, dimethyl sulfoxide, 1,2-propanediol, ethylene glycol, n-dimethylformamide and 1,3-propanediol, as well as acetamide, agarose, alginic acid, alanine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, cyclohexanediol, dextran, diethylene glycol, dimethylacetamide, dimethylformamide, dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glyceryl monoacetate, glycine, Contains hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, mannitol, mannose, methanol, methoxypropanediol, methylacetamide, methylformamide, methylurea, methyl glucose, methylglycerol, phenol, pluronic polyol, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, propanediol, pyridine N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine, and xylose.

[0048] Other cryoprotectants that may be used are described in International Publication No. 02 / 32225, corresponding to U.S. Patent No. 6,395,467 to Fahy et al., U.S. Patent No. 6,274,303 to Wowk et al., U.S. Patent No. 6,194,137 to Khirabadi et al., U.S. Patent No. 6,187,529 to Fahy et al., U.S. Patent No. 5,962,214 to Fahy et al., U.S. Patent No. 5,955,448 to Calaco et al., U.S. Patent No. 5,629,145 to Meryman, and / or U.S. Patent No. 6,740,484 to Khirabadi et al.

[0049] In some embodiments, prior to forming the at least one first biotechnological construct or natural tissue of the present disclosure, the method of the present disclosure may further include immersing the at least one biotechnological construct or natural tissue in a pre-cryopreservation solution (e.g., including one or more of the aforementioned agents and / or cryoprotectants) for a predetermined duration, such as at least 4 hours, or at least 6 hours, or at least 12 hours, or a predetermined duration in the range of 3 to 15 hours, or a predetermined duration in the range of 6 to 12 hours, or a predetermined duration in the range of 8 to 10 hours, or a predetermined duration of about 9 hours, etc. In some embodiments, the pre-cryopreservation solution may include or contain agents selected from the group consisting of antioxidants and caspase inhibitors. Such agents and / or the aforementioned agents and / or cryoprotectants may be present in the pre-cryopreservation solution at concentrations of 1 pM to 2000 mM, 10 pM to 1000 mM, 1 nM to 100 mM, 100 nM to 0.25mM、1μM~1mM、250μM~1mM、1pM~1000μM、1pM~500μM、1pM~30μM、1pM~1000nM、1pM~500nM、1pM~250nM、100pM~750μM、100pM~500μM、100pM~20μM、100pM~1000nM、1pM~750nM、1pM~500nM、1pM~250nM、1pM~1nM、500pM~500μM、500pM~250μM、500pM~100μM、500pM~10μM、500pM~1000nM、500pM~750nM、500pM~500nM、500pM~250nM、500pM~100nM、500pM~1nM、1nM~1000μM、1nM~750μM、1nM~500μM、1nM~250μM、1nM~100μM、1pM~1μM、100nM~1000μM、100nM~750μM、100nM~500μM、100nM~250μM、100nM~100μM、100pM~1μM、250nM~1000μM、250nM~750μM、250nM~500μM、250nM~250μM、250nM~100μM、250nM~1μM、500nM~1000μM、500nM~750μM、500nM~500μM、500nM~250μM、100nM~100μM、500nM~1μM、750nM~1000μM、750nM~750μM、750nM~500μM、750nM~250μM、750nM~100μM、750nM~1μM、0.5μM~1000μM、10μM~950μM、20μM~900μM、30μM~850μM、40μM~800μM、50μM~750μM、60μM~700μM、70μM~650μM、80μM~600μM、90μM~550μM、100μM~500μM、110μM~450μM、120μM~400μM、130μM~350μM、140μM~300μM、150μM~250μM、160μM~200μM、0.The concentrations may be within the ranges of 5 μM to 100 μM, 1 μM to 90 μM, 5 μM to 90 μM, 10 μM to 85 μM, 10 μM to 75 μM, 20 μM to 85 μM, 20 μM to 65 μM, 30 μM to 70 μM, 30 μM to 50 μM, 40 μM to 80 μM, or 40 μM to 50 μM (combinations or individual drugs), where any concentration within the above ranges can also function as the endpoint of the range.

[0050] The volume of the solution employed in the methodology of this disclosure can vary considerably depending on the size of the biomaterial (e.g., natural and / or bioengineered tissues such as bioengineered components).

[0051] In embodiments, the solution comprises a cryoprotectant in an aqueous solution, such as Euro-Collins solution, sterile water, saline solution, culture medium, and any physiological solution.

[0052] The final concentration of cryoprotectant in a solution used for preserving biomaterials (or biotechnically produced biomaterials) (e.g., natural and / or biotechnically produced biomaterials) may be any desired predetermined value, but generally it will be about 70% by weight or less of cryoprotectant (of the total weight of the preservation solution), for example, about 65% by weight or less of cryoprotectant, or about 60% by weight or less of cryoprotectant. In some embodiments, the final concentration of cryoprotectant in a solution used for preservation may be in the range of about 50 to about 80% by weight of cryoprotectant, or about 60 to about 75% by weight of cryoprotectant, or about 68 to about 72% by weight of cryoprotectant (of the total weight of the preservation solution). However, in some embodiments (e.g., biotechnically produced cartilage), the final concentration of cryoprotectant in a solution used for preservation may be higher than the above concentrations.

[0053] In the embodiment, the biomaterial to be preserved (e.g., natural and / or biotechnically engineered tissues such as biotechnically engineered components) may or may not have been previously exposed to a cryoprotectant.

[0054] In embodiments, the biomaterial(s) to be preserved (e.g., bioengineered tissues such as natural tissues and / or bioengineered components) may be immersed in (or exposed to) a solution in which the concentration of the cryoprotectant in the solution may be gradually increased, for example by using a linear or nonlinear concentration gradient to achieve a predetermined final solution cryoprotectant concentration, e.g., ≤70% by weight or less (with respect to the solution flowing through the holes of the cassette of the Disclosure to come into contact with the biomaterial(s)). In such embodiments, the concentration gradient is a linear or nonlinear concentration gradient in which a cryoprotectant-free solution (e.g., a cryoprotectant-free solution initially present in the cassette of the Disclosure and in contact with the biomaterial(s) constituting the cassette) is gradually replaced by a desired solution, such as a solution having a cryoprotectant concentration of ≤70% by weight.

[0055] For example, a cryoprotectant-free solution (e.g., initially present in the cassette of this disclosure) may be substantially replaced with a predetermined solution, such as a solution having a cryoprotectant concentration of ≤70 wt%, over a time period of about 30 minutes, for example, about 10 minutes, or about 5 minutes. In embodiments, the rate at which the cryoprotectant-free solution is replaced with the predetermined solution, such as a solution having a cryoprotectant concentration of ≤70 wt%, should be low enough not to kill most or all of the living cells present, and such a rate depends on the specific tissue / cells of the biomaterial(s) (e.g., natural tissues and / or biotechnical tissues such as biotechnical components) and the size of the individual biomaterial(s). In certain embodiments, the concentration change during vitrification is slow enough to achieve approximate osmotic equilibrium. In other embodiments, the concentration change during vitrification is more rapid so that approximate osmotic equilibrium is not achieved until the final concentration is reached.

[0056] In embodiments, the concentration of the solution is increased stepwise to achieve a predetermined cryoprotectant concentration solution, such as a solution having a cryoprotectant concentration of 70% by weight or less. For example, in embodiments, the concentration of the cryoprotectant may be added stepwise to achieve a specific plateau (e.g., measured in the cassette of the Disclosure), which may be maintained for a predetermined time, for example, a predetermined time in the range of 3 to 10 minutes, or a predetermined time in the range of 4 to 6 minutes, or a predetermined time of about 5 minutes. In certain embodiments, the concentration of the cryoprotectant may be added stepwise to achieve a specific plateau, which may be maintained for a time sufficient to achieve approximate osmotic equilibrium, such as 5 minutes or more, or about 10 minutes or more, or about 15 minutes or more. Next, the cryoprotectant concentration can be increased by adding further cryoprotectant to the first cryoprotectant solution, or a second solution of more concentrated cryoprotectant can be substituted for the first cryoprotectant solution. Then, after maintaining the concentration for a predetermined time (e.g., corresponding to the above) or a time sufficient to achieve approximate osmotic equilibrium, additional cryoprotectant may be added or replaced with a more concentrated form in one or more steps to achieve a desired concentration, e.g., a cryoprotectant concentration of ≤70% by weight.

[0057] In embodiments, there may be any number of cryoprotectant concentration plateaus and / or steps, such as any integer between 2 and 10, before reaching a desired concentration, such as ≤70% by weight of the cryoprotectant. For example, in embodiments, four cryoprotectant concentration plateaus may be used before reaching a desired concentration, such as ≤70% by weight of the cryoprotectant.

[0058] In some embodiments, there may be six steps, the first step using a solution without a cryoprotectant, followed by four increasing plateaus of cryoprotectant concentrations, and then a final predetermined cryoprotectant concentration, for example, ≤70% by weight. For example, in such embodiments where the final predetermined cryoprotectant concentration is about 70% by weight of cryoprotectant, step 1 may use no cryoprotectant, step 2 may use about 5 to about 20% of the final cryoprotectant concentration, for example, about 10 to about 15%, step 3 may use about 15 to about 35% of the final cryoprotectant concentration, for example, about 20 to about 30%, step 4 may use about 40 to about 60% of the final cryoprotectant concentration, for example, about 45 to about 55%, step 5 may use about 65 to about 85% of the final cryoprotectant concentration, for example, about 70 to about 80%, and step 6 may use a final cryoprotectant concentration of about 70% by weight of cryoprotectant. In some embodiments, each cryoprotectant concentration step may be for a predetermined duration, for example, a predetermined duration in the range of 3 to 10 minutes, or a predetermined duration in the range of 4 to 6 minutes, or a predetermined duration of about 5 minutes. In some embodiments, each cryoprotectant concentration step may be maintained for a time sufficient to achieve approximate osmotic equilibrium.

[0059] For example, in some embodiments, the at least one biotechnological component or natural tissue sample is immersed in 1 to 6 different solutions, or a series of solutions in which the concentration of the cryoprotectant decreases is obtained via a linear or nonlinear concentration gradient. In some embodiments, the at least one biotechnological component or natural tissue sample is immersed in the 1 to 6 different solutions, and the at least one biotechnological component or natural tissue sample is immersed in each of the different solutions for 5 minutes or less.

[0060] In some embodiments, the method of the present disclosure includes the steps of (i) immersing the at least one biotechnological component or natural tissue sample in a series of solutions with increasing concentrations of cryoprotectant to form at least one first biotechnological component or natural tissue immersed in a final solution having a cryoprotectant concentration of 70% by weight or less; (ii) cooling the at least one first biotechnological component or natural tissue in the final solution having a cryoprotectant concentration of 70% by weight or less to a temperature below the glass transition temperature of the final solution having a cryoprotectant concentration of 70% by weight or less; and (iii) immersing the at least one first biotechnological component or natural tissue in a series of solutions with decreasing concentrations of cryoprotectant. The method comprises the steps of immersing a sample of the at least one first biotechnological component or natural tissue in a solution substantially free of cryoprotectants, wherein the at least one second biotechnological component or natural tissue is a substantially cryoprotectant-free component, and in step (iii), immersing the sample of the at least one first biotechnological component or natural tissue in 1 to 7 different solutions (and immersing the sample of the at least one first biotechnological component or natural tissue in each of the different solutions for 5 minutes or less), or obtaining a series of solutions with increasing concentrations of the cryoprotectant via a linear or nonlinear concentration gradient.

[0061] A biomaterial (e.g., a bioengineered tissue such as a natural tissue and / or a bioengineered component) can be rapidly cooled (preferably at a rate in the range of about 35°C / min to about 55°C / min, or at a rate of about 45°C / min) to a temperature between about -20°C and the glass transition temperature (e.g., while the biomaterial(s) are contained in the cassette of the Disclosure), or to a temperature of about -100°C. In some embodiments, the rapid cooling rate can be about -15 to about -75°C per minute. For example, the average cooling rate can be about -15 to about -75°C per minute, e.g., about -30 to -60°C per minute, or about -35 to -50°C per minute, or about -43 to -47°C per minute. During this rapid cooling process, the temperature at which the biomaterial(s) are cooled is between approximately -20°C and the glass transition temperature of a predetermined final cryoprotectant solution where the cryoprotectant concentration is 70% by weight or less, for example, between approximately -80°C and approximately -180°C, or between approximately -90°C and approximately -120°C, or approximately -100°C.

[0062] Biomaterials(s) (e.g., natural and / or biotechnical tissues such as biotechnical components) may also optionally undergo a slow cooling step after a rapid cooling step (e.g., while the biomaterials(s) are configured in the cassette of the disclosure), in which the biomaterials(s) may be cooled to a predetermined storage temperature above the glass transition temperature at an average rate of less than 30°C per minute, for example, at an average rate of less than 10°C per minute. The cooling step may be carried out at an average rate of less than 5°C per minute, or less than about 3°C ​​per minute. In embodiments, the cooling rate during the entire slow cooling step is a cooling rate that does not increase by more than 30°C per minute, for example, a cooling rate that does not increase by more than 10°C per minute, or a cooling rate that does not increase by more than 5°C per minute. In embodiments, the cooling rate (for a single or multi-stage cooling process) includes, for example, a cooling rate in the range of about 0.5 to about 10°C / min, for example, about 2 to about 8°C / min, or about 4 to about 6°C / min. In the embodiment, the process is independent of the cooling rate, as long as ice formation is avoided. The temperature at which the biomaterial(s) are cooled during this slow cooling process is between approximately -110°C and approximately -180°C, or approximately -125°C and approximately -145°C, or approximately -135°C.

[0063] In this embodiment, a slow cooling rate is achieved by changing the environment in which the container containing the solution is placed.

[0064] In some embodiments, a rapid cooling rate is achieved with the help of an additional liquid, such as 2-methylbutane, which is optionally pre-cooled. Then, to achieve a slow cooling rate, the container (e.g., the cassette of this disclosure) is removed from the liquid and further cooled in a gaseous environment to the final storage temperature.

[0065] Biomaterials(s) (e.g., natural and / or biotechnological tissues such as biotechnological components) can be stored for a predetermined period at a temperature below -20°C and below the glass transition temperature (e.g., while the biomaterials(s) are configured within the cassette of this disclosure). For example, after the cooling step described above, the biomaterials(s) can be stored at a temperature between approximately -110°C and approximately -180°C, or between approximately -125°C and approximately -145°C, or at approximately -135°C.

[0066] In some embodiments, the method may further include a transport step in which biomaterials (e.g., natural and / or biotechnically engineered tissues such as biotechnically engineered components) are transported (e.g., while the biomaterial(s) are contained in the cassette of the Disclosure). In embodiments, the biomaterial(s) are transported at a temperature between the glass transition temperature of the final maximum strength solution and -20°C, for example, about 20°C to 80°C higher than the glass transition temperature of the maximum strength cryoprotectant solution, and 40°C to 60°C higher than the glass transition temperature of a given maximum strength cryoprotectant solution, such as a solution having a cryoprotectant concentration of ≤70 wt%, or a solution having a cryoprotectant concentration of ≤70 wt%. For example, the biomaterial(s) can be transported on dry ice at about -79.6°C.

[0067] After storage, the biomaterial(s) (e.g., natural tissues and / or biotechnically engineered tissues such as biotechnically engineered components) can be removed from a predetermined maximum-intensity cryoprotective solution. A method for removing the biomaterial(s) from a predetermined maximum-intensity cryoprotective solution may include slowly warming the biomaterial(s) in the predetermined maximum-intensity cryoprotective solution to a warming temperature in the range between -20°C and the glass transition temperature of the cryoprotective solution (e.g., while the biomaterial(s) are configured in the cassette of this disclosure). A slow warming rate of less than 50°C per minute may be used to warm the biomaterial(s) in the predetermined maximum-intensity cryoprotective solution. In embodiments, the average warming rate during this stage may be about 10 to 40°C per minute, for example, about 25 to 35°C per minute. Furthermore, the temperature to which the stored biomaterial(s) are slowly warmed may be about -30°C to -80°C, for example, about -45°C to -65°C.

[0068] After the biomaterial(s) (e.g., natural and / or biotechnically engineered tissues such as biotechnically engineered components) have undergone this optional slow heating process, the biomaterial(s) may then be rapidly heated to a temperature above -20°C (e.g., while the biomaterial(s) are configured within the cassette of this disclosure). In embodiments, the temperature should be sufficiently high so that the solution is sufficiently fluid so that the biomaterial(s) can be removed from it. The rapid heating process may be carried out at a rate above about 80°C per minute, for example, above about 100°C per minute. The average heating rate during this step may be about 200–300°C per minute, for example, about 215–250°C per minute. In embodiments, the biomaterial(s) may be heated to a temperature above about -20°C, for example, above about -10°C, or above about -5°C, for example, between about -5°C and about 5°C. In embodiments, the process is independent of the heating rate, as long as ice formation is avoided.

[0069] In embodiments, a rapid heating rate can be achieved by changing the environment in which the container containing the solution is placed. In embodiments, a slow heating rate can be achieved by placing the container (e.g., the cassette of the present disclosure) in a gaseous environment at a temperature above the temperature in which the biomaterial(s) (e.g., natural tissues and / or biotechnical tissues such as biotechnical components) are stored. Then, to achieve a rapid heating rate, the container can be placed in a coil of an induction heating system or in a liquid, such as an aqueous solution of dimethyl sulfoxide (DMSO), at a temperature above -75°C, for example above 0°C, or at normal ambient temperature.

[0070] In embodiments, after a biomaterial(s) (e.g., natural and / or biotechnological tissues such as biotechnological components) has been heated to a temperature above -65°C, the concentration of the cryoprotectant in the solution can be gradually or stepwise reduced, for example, by reversing the steps described above to increase the concentration of the cryoprotectant (e.g., while the biomaterial(s) are in the cassette of the Disclosure). For example, in embodiments, the biomaterial(s) to which the cryoprotectant concentration is reduced can be immersed in (or exposed to) a solution in which the cryoprotectant concentration of the solution can be gradually reduced to achieve a nearly cryoprotectant-free or cryoprotectant-free solution, for example, by using a linear or nonlinear concentration gradient. In embodiments, the concentration gradient is a linear or nonlinear concentration gradient in which a solution having a cryoprotectant concentration of a predetermined highest strength cryoprotectant solution, for example, a solution having a cryoprotectant concentration of ≤70 wt%, is gradually replaced by a cryoprotectant-free solution.

[0071] In embodiments, the cryoprotectant concentration is progressively reduced (e.g., while the biomaterial(s) (e.g., natural and / or biotechnological tissues such as biotechnological components) is contained in the cassette of the Disclosure). In embodiments, the reduction in the cryoprotectant concentration of the tissue can be achieved by immersing the tissue in a series of solutions with decreasing cryoprotectant concentrations to facilitate the elution of the cryoprotectant from the tissue. The solutions are generally at temperatures above about -15°C, e.g., about -15°C to about 15°C, or about 0°C to about 10°C.

[0072] In some embodiments, the cryoprotectant concentration may be reduced to achieve a specific plateau, which may be maintained for a predetermined period, for example, a predetermined period in the range of 3 to 10 minutes, or a predetermined period in the range of 4 to 6 minutes, or a predetermined period of about 5 minutes. In some embodiments, the cryoprotectant concentration may be reduced to achieve a specific plateau, which may be maintained for a time sufficient to achieve approximate osmotic equilibrium.

[0073] Next, the concentration of the cryoprotectant may be further reduced, thereby providing or not providing a cryoprotectant-free solution. Otherwise, optionally, after maintaining the concentration for a sufficient time to achieve approximate osmotic equilibrium, the concentration of the cryoprotectant can be further reduced again in one or more steps to finally provide a cryoprotectant-free solution. In embodiments, the tissue may be immersed in each solution for a predetermined period, for example, a predetermined period in the range of 3 to 10 minutes, or a predetermined period in the range of 4 to 6 minutes, or a predetermined period of about 5 minutes.

[0074] To reduce the concentration of the cryoprotectant, the cryoprotectant solution may be mixed with a solution of a similar type to those used when adding cryoprotectants to biomaterials (e.g., natural and / or bioengineered tissues such as bioengineered components). The solution may also contain at least one osmotic buffer.

[0075] As used herein, "osmotic buffering agent" means a low-molecular-weight or high-molecular-weight non-osmotic extracellular solute that counteracts the osmotic effect of cryoprotective agents at intracellular concentrations higher than extracellular concentrations during the cryoprotective agent efflux process.

[0076] As used herein, "non-penetrating" means that the majority of the molecules of a chemical substance do not penetrate into the cells of a biomaterial (plural) (e.g., natural tissues and / or bioengineered tissues such as bioengineered components), but instead remain in the extracellular fluid of the biomaterial.

[0077] As used herein, "low molecular weight" refers to a relative molecular weight of, for example, 1,000 daltons or less. As used herein, "low molecular weight osmotic buffering agents" have a relative molecular weight of 1,000 daltons or less. Examples of low molecular weight osmotic buffering agents include maltose, potassium and sodium fructose-1,6-diphosphate, potassium and sodium lactobionate, potassium and sodium glycerophosphate, maltopentose, stachyose, mannitol, sucrose, trehalose, glucose, maltotriose, sodium and potassium gluconate, sodium and potassium glucose-6-phosphate, and raffinose. In embodiments, the low molecular weight osmotic buffering agent is at least one of mannitol, sucrose, trehalose, and raffinose.

[0078] As used herein, "high molecular weight" refers to a relative molecular weight, for example, greater than 1,000 daltons and less than or equal to 500,000 daltons. As used herein, "high molecular weight cryoprotectant and osmotic buffering agents" generally have a relative molecular weight greater than 1,000 daltons and less than or equal to 500,000 daltons. Examples of high molecular weight osmotic buffering agents include hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), raffinose undecaacetate (greater than 1,000 daltons), and Ficol (greater than 1,000 to 100,000 daltons). In embodiments, the high molecular weight osmotic buffering agent is HES, such as HES having a molecular weight of about 450,000.

[0079] A solution without cryoprotectant may contain an osmotic buffer of less than approximately 500 mM, for example, an osmotic buffer of approximately 200-400 mM. A low molecular weight osmotic buffer may be used as the osmotic buffer. In this embodiment, the low molecular weight osmotic buffer is mannitol.

[0080] In embodiments, the cryoprotectant can be removed in a series of steps such as 3, 4, 5, 6, or 7. In embodiments, the cryoprotectant may be removed in a series of 7 steps, in which step 1, the biomaterial(s) (e.g., natural tissues and / or biotechnological tissues such as biotechnological components) may be exposed to a cryoprotectant solution at a concentration that may be about 40 to about 70% of the maximum cryoprotectant concentration used, for example, about 45 to about 55%, and in step 2, the biomaterial(s) may be exposed to a cryoprotectant concentration that may be about 30 to about 45% of the maximum cryoprotectant concentration used, for example, about 35 to about 40%. In step 3, the biomaterial(s) may be exposed to a cryoprotectant concentration of about 15 to about 35% of the maximum cryoprotectant concentration used, for example, about 20 to about 30%; in step 4, the biomaterial(s) may be exposed to a cryoprotectant concentration of about 5 to about 20% of the cryoprotectant concentration used, for example, about 10 to about 15%; and in step 5, the biomaterial(s) may be exposed to a concentration of about 2.5 to about 10% of the cryoprotectant concentration used, for example, about 5 to about 7.5%. In the above steps, the remainder of the solution may contain an osmotic buffer and be a solution without cryoprotectant. In step 6, substantially all of the cryoprotectant may be removed, while the osmotic buffer may be retained. In step 7, the osmotic buffer may be removed. In embodiments, steps 6 and 7 may be combined in a single step. For example, the osmotic buffer may be removed at the same time as the remaining cryoprotectant. In embodiments, if the osmotic buffer is not used or not removed, step 7 may be omitted. Each of these concentration steps may be maintained for a sufficient time to achieve approximate osmotic equilibrium, such as about 10–30 minutes or 15–25 minutes. In some embodiments, each of the concentration steps may be maintained for about 4–6 minutes or about 5 minutes. In embodiments, the cryoprotectant is removed by one or more washes using a cryoprotectant-free solution.

[0081] The temperature of the series of solutions used to remove cryoprotectants from biomaterials (or biotechnically engineered tissues, e.g., natural and / or biotechnically engineered tissues, such as biotechnically engineered components) may be about -15°C or higher, e.g., about -15 to about 15°C, or about 0°C to about 37°C. In embodiments, step 1 may be started when the biomaterials (or biotechnically engineered tissues) are at a temperature above about -75°C, e.g., above -65°C. In embodiments, the temperature of the biomaterials (or biotechnically engineered tissues) may be below the temperature of the solution into which they are immersed in step 1, and the biomaterials (or biotechnically engineered tissues) may be further warmed to a temperature above about -15°C during step 1 of cryoprotectant removal.

[0082] Freeze-free solutions used for washing biomaterials (or biotechnically engineered tissues such as natural tissues and / or biotechnically engineered components) may be sterile water, physiological saline solutions (e.g., saline solution, Hank's Balanced Salt Solution, Lactated Ringers Solution, or Krebs-Henseliet Solution), or tissue culture media used for tissues such as mammalian cells (e.g., Roswell Park Memorial Institute medium, Dulbecco's Modified Eagle's Medium (DMEM), Eagle's Medium, or Medium 199).

[0083] The number of washes, the amount of each wash, and the duration of each wash may vary depending on the mass of the individual biomaterials and the desired final residual chemical concentration. In embodiments, the final wash (rinse) may be performed in a commonly used medical saline solution, such as physiological saline or Ringer's solution.

[0084] In some embodiments, the methodology of the present disclosure may be carried out in vials or deep-well plates. Alternatively, the methodology of the present disclosure may be carried out in specially designed cassettes of the present disclosure, which are fabricated for use in vitrification processes. The cassettes of the present disclosure can be used as part of a high-capacity system capable of storing multiple biomaterials (e.g., natural and / or biotechnological tissues such as biotechnological components) at once.

[0085] The biggest hurdle when preserving multiple components at once is the ability to generate and maintain appropriate cooling and reheating rates that prevent ice formation. Previous research related to cryopreservation of cells on plates has shown that the system configuration significantly impacts the ability to cool and heat without ice formation. Initially, it was thought that vitrification of components could be performed using multi-well culture plates (similar to those in which components are typically shipped to end users).

[0086] However, the design of such plates makes them difficult to use for low-temperature (i.e., cryogenic) storage. Conventional multi-well culture plate designs make it difficult to place them in a cooler and warmer without the contents of the cooler and warmer flowing into the wells. Deep-well plates can accommodate larger volumes and are designed to reduce the risk of wicking (core formation) in the wells.

[0087] The methodology described above was used to successfully vitrify six components at once using deep-well plates. However, vitrification of more than six components at once was shown to result in reduced viability due to prolonged exposure to cytotoxic CPAs.

[0088] In embodiments, deep-well plates for use with the methodology of this disclosure should be made from a different plastic (i.e., polypropylene compared to polystyrene) compared with conventional tissue culture plates. Polypropylene has proven to be more susceptible to vitrification and low-temperature effects by providing better conductivity for cooling and heating biomaterials (e.g., natural and / or biotechnically engineered tissues such as biotechnically engineered structures). More consistent and faster cooling and heating rates were achieved.

[0089] In some embodiments, the methodology described above in the Disclosure may be carried out in a specially designed cassette of the Disclosure, which is adapted for use in a vitrification process. The cassette of the Disclosure may be used as part of a high-capacity system capable of storing multiple biomaterials (e.g., natural and / or biotechnological tissues such as biotechnological components) at once.

[0090] For example, the methodology described herein may be carried out in a cassette designed to hold 24 biomaterial samples (e.g., natural and / or bioengineered tissues such as bioengineered components) in place within a well insert, while providing sufficient space and access for the biomaterials to be exposed to the vitrification solution. The cassette is configured to move as a single unit, thus reducing the time between steps and making the injection / removal steps more seamless. Furthermore, the configuration of the inserts within the cassette can be set to mimic the configuration of the wells in a 24-well plate, so that the deposition of the inserts into the cassette and their return to the plate after reheating can also be performed seamlessly, without requiring the handling of individual biomaterials (this is particularly advantageous compared to protocols where each biomaterial needs to be moved individually, limiting the number of individual biomaterials that can be vitrified at one time). The cassette fragments can be easily modified for other types of native / natural tissues and / or biotechnological components, and are very suitable for use in automated settings where multiple biomaterial samples (in this specification, the terms biomaterial(s) and biomaterial sample(s) are used interchangeably) can be processed, vitrified, and reheated as a single unit.

[0091] Such a cassette may have two parts (e.g., upper and lower) that interlock to hold a predetermined number of well inserts (the exemplary embodiments depicted and described below have 24 well inserts, but the predetermined number of well inserts may range from 6 to 384, such as 12 to 192, or 24 to 96, or 24 to 48). The upper part has a placeholder for holding the inserts in place in a configuration that fits a predetermined well arrangement, such as the well arrangement of a 24-well plate. This configuration facilitates the transfer of each biomaterial sample from the cassette to a multi-well plate as needed. Holes may be present in both parts of the cassette to allow the vitrification solution to flow easily through and around each insert and biomaterial, so that the cassette can be immersed in the vitrification solution during the CPA injection / removal step and the actual vitrification. In embodiments, the placeholder can be easily modified to also hold fragments of native tissue for preservation.

[0092] In embodiments, the cassette may be configured so that the entire vitrification process can be automated. The cassette of this disclosure, capable of holding a large number of biomaterial samples at once (e.g., natural and / or bioengineered tissues such as bioengineered components) (e.g., up to 384 biomaterials at once, or 24 biomaterials at once as described below), enables the processing of multiple commercially available samples in a reproducible manner. The use of the cassette of this disclosure enables improvements to the vitrification method by reducing handling errors and improving product quality, while making it practical to process batches of components and / or tissues with a higher level of precision, while ensuring a high level of viability for each specimen for banking.

[0093] An exemplary cassette of the present disclosure is shown in Figure 1 below. Figure 1 shows an exemplary cassette (100) having a first part (102) and a second part (104) configured to interlock with each other via a releasable locking mechanism, wherein one or more spaces in the side wall (118) of the second part are configured to receive one or more protruding structures (106) of the second part (104). Holes (108, 116) are present in both the first part (102) and the second part (104) of the cassette (100) to allow fluids (e.g., vitrification solution or solutions used during the vitrification process) to flow easily through the cassette (100). The second part (104), which may be at the top of the cassette (100), has a placeholder (110) for holding an insert (see (120) in Figure 2) in place in a configuration that conforms to the well arrangement of a well plate (not shown). This configuration facilitates the transfer of biological samples from the cassette to the plate as needed.

[0094] Figure 2A shows an exemplary cassette as a one-piece unit, illustrating how the biomaterial (122) in the insert (124) fits into the cassette. Figure 2B is a side view of the cassette in the closed position. Figure 2C shows a side view of the cassette with the lid removed. Figure 2D illustrates two sides of the cassette with the biomaterial sample positioned on one side, and Figure 2E illustrates side views of both sides of the cassette.

[0095] As shown in Figures 2A and 2D, holes (108, 116) are present in both the first portion (102) and the second portion (104) of the cassette (100), allowing fluid (e.g., vitrification solution or solution used during the vitrification process) to flow easily through the cassette and around each insert (124) and biomaterial (122).

[0096] Figure 3 is a photograph of an exemplary cassette showing the relative relationship of its parts. This cassette has two parts, both of which have holes to allow the solution to easily reach the biomaterial. The lid includes a round placeholder to hold the well insert containing the biomaterial in place within the cassette.

[0097] In some embodiments, the cassettes of this disclosure can be made from plastics conventionally used in the manufacture of laboratory equipment for research and medical purposes, particularly plastic formulations that can withstand exposure to chemicals used in cryogenic and vitrification processes. In some embodiments, the plastic may be polypropylene or Teflon.

[0098] The cassettes of this disclosure may be reusable or disposable.

[0099] The cassettes of this disclosure may be manufactured using conventional methods, such as injection molding.

[0100] The cassettes of this disclosure can be manufactured using a reaction injection molding technique, which involves injecting a prepolymer into a mold instead of using a molten polymer material. After injection, the prepolymer polymerizes and hardens to form the finished portion of the cassette of this disclosure. In addition, since prepolymers generally have lower viscosity than molten polymers, they flow more easily into the mold, which can reduce mold manufacturing costs.

[0101] In some embodiments, the cassette of the present disclosure may be a reusable or disposable multi-sample cassette comprising a first part and a second part configured to fit together via a releaseable locking mechanism, the second part comprising one or more inserts and a plurality of placeholders, each placeholder of the plurality of placeholders configured to receive one of the one or more inserts, each of the one or more inserts comprising at least one biotechnological component or natural tissue sample, the first part and the second part comprising a plurality of openings configured to allow a solution to flow through and around each of the one or more inserts and at least one biotechnological component or natural tissue sample, the configuration of the placeholders in the cassette being set to mimic the configuration of wells in a plate having a predetermined number of wells, the predetermined number of wells ranging from 6 to 384.

[0102] In embodiments, the releasable locking mechanism may be one of those depicted above, and / or a mechanism in which engagement of the releasable locking component depends on an initial spatial deflection of at least one element of the locking component. These may be, for example, bayonet tabs with a return stopper that engages with a coupling element of the other locking component, followed by a spatial re-deformation of at least one element to catch on one or more elements of the other locking component. The anti-rotation and coupling components are adapted to be releasable by making the anti-rotation relatively small so that the force required to disengage from the coupling form is small. The coupling component may be a bar or the edge of an opening from which the bayonet tab can protrude.

[0103] Alternative methods for releasably connecting the first and second parts include, for example, the use of a ball and socket, where the ball component mates with a socket slightly smaller than the diameter of the ball. Similar to a tab and detent system, manual force is applied to push the ball into the socket, causing one or both parts to deform and pass over each other to a first locked position. The locking elements can be positioned along the outer edges of the first and second parts. For large reusable or disposable multi-sample cassettes (e.g., including 384 wells), multiple large locks can be positioned along each side to help maintain a secure locking state between the first and second parts. In some embodiments, when the releasable lock is moved to its final locked position with the second and third locking elements engaged, a space is formed between the engaged locking elements and the lid or tray. This space can be resized by appropriate sizing of the locking elements and / or to provide a handle for manual handling of the reusable or disposable multi-sample cassette.

[0104] In an embodiment, the first portion may have a frame having a predetermined thickness, such as about 0.5 to about 3 mm, or about 1 to about 2 mm, or about 1.3 to about 1.8 mm, or about 1.4 to about 1.6 mm. In an embodiment, the second portion may have a frame having a predetermined thickness, such as about 0.5 to about 3 mm, or about 1 to about 2 mm, or about 1.3 to about 1.8 mm, or about 1.4 to about 1.6 mm.

[0105] In an embodiment, each of the plurality of openings (which may be in the form of any desired shape such as circular or hexagonal) may have a diameter in the range of about 3 to about 7 mm, or about 4 to about 6 mm, or about 4.5 to about 5.5 mm, or about 4.8 to about 5.2 mm. Additionally, each of the plurality of openings may be spaced apart by a distance within a predetermined range, for example, 3 to 15 mm, or about 5 to about 12 mm, or about 7 to about 10 mm, or about 8 to about 9 mm from the nearest adjacent opening. In some embodiments, the diameter of each of the plurality of openings is the same in both the first portion and the second portion. In other embodiments, the diameter of each of the plurality of openings in the first portion is different from the diameter of each of the plurality of openings in the first portion.

[0106] In some embodiments, the area inside the cassette (when the first and second portions are engaged) is about 89 cm 2 ~ about 101 cm 2 or about 92 cm 2 ~ about 98 cm 2 or about 94 cm 2 ~ about 96 cm 2 or about 94.5 cm 2 ~ about 95.5 cm 2 and may be set to a predetermined area, such as an area within the range.

[0107] In some embodiments, the volume of the entire internal chamber of the reusable or disposable multi - sample cassette (when the first and second portions are engaged) is about 81 cm 3 ~ about 98 cm 3 or about 86 cm 3 ~ about 95 cm 3 or about 89 cm 3 ~ about 92 cm 3 and may be within the range.

[0108] In some embodiments, the volume of each biotechnological construct or natural tissue sample included in the cassette is about 1 cm 3 ~ about 1.5 cm 3 or about 1.1 cm3 ~approx. 1.4cm 3 , or approximately 1.1 cm 3 ~approx. 1.3cm 3 It may be within a certain range. Such one biotechnological component or natural tissue sample can be fixed / rested on the surface of the one or more inserts by conventional methods known to those skilled in the art.

[0109] In some embodiments, the first and / or second parts may be disposable or reusable. In this regard, the first and / or second parts, or any part thereof, may be made from, but are not limited to, polyolefins such as polyethylene and / or copolymers thereof including low-density, high-density, linear low-density, or ultra-low-density polyethylene, polypropylene copolymers including polypropylene and / or atactic polypropylene, isotactic polypropylene, syndiotactic polypropylene, and / or combinations thereof, or from plastic materials such as polybutylene. In other embodiments, the disposable or reusable first and / or second parts may be made from glass or ceramic materials, etc.

[0110] In some embodiments, the first and second parts may be formed from a medical-grade plastic material such as medical-grade polypropylene, polystyrene, or Teflon. The first and second parts may be formed from a transparent or translucent plastic material, or may include transparent or translucent portions (made of medical-grade polypropylene, polystyrene, Teflon, or other plastics) so that a user or operator can observe one or more biomaterials contained therein. In such embodiments, only selected portions of the first and / or second parts may be transparent or translucent, while the other portions of the first and second parts are formed from an opaque reflective material.

[0111] In embodiments, the Disclosure provides a device (such as a perfusion device) for supplying the above-mentioned solution to a cassette of the Disclosure. The perfusion device includes at least one solution source well that is in fluid communication with an internal chamber of the cassette of the Disclosure containing biomaterial (e.g., bioengineered tissue such as natural tissue and / or bioengineered constructs) and controlsly provides a source of each solution to a fresh culture medium in one chamber containing the biomaterial(s), and a waste well that is in fluid communication with the chamber of the cassette and controlsly receives waste culture medium and / or fluids flowing out of the cassette.

[0112] The specific and preferred values ​​disclosed for components, raw materials, additives, dimensions, conditions, and similar embodiments, as well as their ranges, are illustrative and do not preclude other defined values ​​or other values ​​within the defined ranges. The apparatus and methods of this disclosure may include any value or any combination of the values, specific values, more specific values, and preferred values ​​described herein, including express or implicit intermediate values ​​and ranges. [Examples]

[0113] [method] <Tissue culture> A commercially available human epidermal model was obtained and maintained according to its specifications. All components were incubated for 24 hours in appropriate medium under physiological tissue culture conditions before starting the experiment. A fresh control was used in each experiment, and the experimental treatment groups were evaluated over 4-5 days after re-warming.

[0114] <Vitrification process> To achieve the final cryoprotection agent concentration, each formulation was gradually impregnated with a vitrified solution pre-cooled at 4°C in six stages: 0%, 12.5%, 25%, 50%, 75%, and 100%. After reheating, the vitrified solution was sequentially removed into the culture medium in seven stages at 4°C as described above. (See Non-Patent Literature 15: Vitreous cryopreservation maintaining the function of vascular grafts, Nature Biotechnology, 8(3):296-9, Epub 2000 / 03 / 04, doi:10.1038 / 73737, PubMed PMID: 10700144 (2000); Song et al., Vitreous Preservation of Rabbit Articular Cartilage, Cell Preservation Technology, 2 (1); 67-74 (2004)).

[0115] Once the vitrification solution has been filled into the container, it can be cooled to the storage temperature and reheated using several methods. (1) The container was placed in a glass scintillation vial (diameter × height, 25 mm × 60 mm), 1.5 mL of pre-cooled vitrification solution was added, and 0.3 mL of the solution was placed in the well insert. Next, 1 mL of 2-methylbutane (isopentane, freezing point: -160°C, density: 0.62) was placed on top of the vitrification solution in the vial, and 0.2 mL was placed in the well insert at 4°C, ensuring that it was not directly exposed to air. The sample was placed in a pre-cooling bath containing isopentane in a mechanical storage freezer at -135°C and rapidly cooled to -100°C (approximately 45°C / min). Once -100°C was reached, the sample was removed from the bath and stored at -135°C in a mechanical storage freezer that cooled slowly to -135°C (3°C / min). The sample was kept at -135°C for a minimum of 24 hours. The components were reheated in two stages. First, they were slowly heated to -100°C (approximately 30°C / min) at the top of a mechanical storage freezer, and then rapidly heated to 0°C or ±-10°C (approximately 225°C / min) in a 30% ME2SO bath at room temperature. (2) The components were inserted into the wells of the deep-well plate. 0.6 mL of vitrification solution was placed in each well, and 0.2 mL of solution was placed in each insert. To cool the plate, it was placed in a shallow pre-cooling bath containing isopentane in a mechanical storage freezer at -135°C for approximately 5 minutes. After that, the plate was removed from the bath and left in the mechanical storage freezer at -135°C to cool slowly to -135°C for storage. For reheating, the plate was removed from the freezer and left at room temperature to slowly reheat the sample to approximately -100°C. Then, the deep-well plate was rapidly reheated by placing it in a 30% ME2SO bath at room temperature until the sample no longer vitrified. (3) The last method is to place the components into an insert and then into a specially made cassette. Once the loading step is complete, the cassette is placed in a pouch containing approximately 150 mL of vitrification solution and sealed by removing the air with a bag sealer. The bag is then placed in a pre-cooling bath containing isopentane overnight in a mechanical storage freezer at -135°C. The next day, the bag is removed from the bath and stored at -135°C.To reheat the sample, the bag containing the cassette is placed at -80°C for 15-25 minutes to allow it to cool slowly. Then, the bag is rapidly heated by immersing it in a water bath at approximately 40°C until the sample no longer vitrifies.

[0116] <Viability (survival rate) assay> (Resazurin assay) Used to measure metabolic activity, and being non-toxic, it has the advantage of allowing evaluation of components several times before and after treatment. Resazurin dye (alamarBlue) was used to assess cell viability by measuring the oxidation / reduction reaction occurring within cells. The dye was added directly to the culture wells, and the plates were incubated at 37°C for 3 hours. Upon reduction, the dye changed color and was measured and quantified using a fluorescence microplate reader at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

[0117] (MTT assay) It was also used to measure metabolic activity. This assay was included because it is the most common assay used to assess skin equivalent viability. The MTT[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay is based on the ability of mitochondrial dehydrogenase enzymes from viable cells to cleave the tetrazolium ring of pale yellow MTT and form deep blue formazan crystals that accumulate in healthy cells. When cells are solubilized with detergent, the solubilized crystals are released. The number of viable cells is directly proportional to the level of formazan product produced and can be quantified using a simple colorimetric assay read with an absorbance reader.

[0118] (Dose-response assay) (Functional endpoint assay required by MatTek for quality assurance) The dose-response assay was performed according to the manufacturer's instructions. Fresh tissues were used in the Triton Dose Response assay the next day, and the vitrified group was vitrified the next day. The test starts by applying 100 μl of 1% Triton X-100 at time intervals of 4, 6, 8, and 12.5 hours. After Triton exposure, the tissue constructs are washed with sterile PBS and immediately transferred to the MTT assay (see below) used to evaluate the cell viability of the constructs. The assay meets the acceptance criteria if the ET-50 or 50% viability decreases within 4.77 - 8 hours for epidermal tissue and 12.2 < ET-50 < 37.5 minutes for EpiOcular tissue.

[0119] (IL-1α release) IL-1α is an important regulator of immune and inflammatory responses. In addition to the MTT assay, IL-1α is used to measure and predict the irritancy of substances tested using an epidermal model. Since IL-1α is released into the supernatant, samples from fresh and cryopreserved epidermal constructs are stored, and IL-1α is measured using an EIA assay.

[0120] The first vitrification protocol was developed for use with vein segments and rings. This process uses a 6-step (15-minute incubation) protocol to add a cryoprotectant (CPA) solution to the constructs. Then, the inserts are placed into glass scintillation vials, and 1.5 mL of vitrification solution and 0.3 mL of solution are placed inside the inserts. The vials are rapidly cooled to -100 °C and then slowly cooled to -135 °C and stored until rewarming. During rewarming, the samples are slowly warmed to -100 °C and then rapidly warmed to room temperature. The vitrification solution is removed using 7 consecutive removal steps of 15 minutes each (Figure 4, original protocol). Several modifications were made to this protocol to obtain a better and more sustainable engraftment rate of epidermal constructs. Some of these modifications are shown in Table 1.

[0121]

Table 1

[0122] The simplest adjustment was to change to different vitrification solutions for the containers with varying VS55–VS70 concentrations, each containing different amounts of the same components: dimethyl sulfoxide (DMSO), propanediol (PD), and formamide (FD) (Figure 4, modified protocol).

[0123] A series of modifications were made to the method of injecting and removing the vitrification solution from the components. These modifications included shortening the incubation step (5 minutes instead of 15 minutes), using a low concentration of cryoprotectant for injecting the highest-intensity vitrification solution into the components, and more mechanical changes, such as diluting the highest-intensity vitrification solution to half its concentration by a simple dilution step, as opposed to removal from one solution to the next. This reduced the time the components were exposed to the highest-intensity vitrification solution and mitigated the potential cytotoxicity caused by exposure to the vitrification solution (modified protocol). Finally, the addition of the antioxidant α-tocopherol (αT) and the caspase inhibitor Q-VD-OPH (QVD) to the culture medium of the components before and after vitrification improved viability and also improved the maintenance of viability for several days after rewarming (Figure 1, added additives).

[0124] Other additives were also tested, but only QVD and αT showed significant improvement.

[0125] The protocol begins with an overnight incubation using αT and QVD. The following day, the components are vitrified using a 6-step addition protocol with each step lasting 5 minutes, in order to add cryoprotectant (CPA) solution to the components. The inserts are then left in the final vitrification solution containing 0.3 mL of the solution within the inserts. The vials are rapidly cooled to -100°C, then slowly cooled to -135°C, and stored until re-warming. During re-warming, the samples are slowly warmed to -100°C, then rapidly warmed to room temperature. The vitrification solution is removed via seven consecutive removal steps of 5 minutes each. In the first removal step, the final vitrification solution is diluted to 50% of its final concentration. The components are left in culture medium + additives for at least 24 hours after re-warming to promote viability for at least 2-3 days after re-warming.

[0126] Having established protocols more suitable for these 3D structures, we conducted further searches for the optimal vitrification solution. While several issues related to the sustained viability of the structures were overcome, further improvements in viability, such as maintaining viability for several days after re-warming, were desired.

[0127] Cytotoxicity was a major concern because the vitrification solution had a high concentration of cryoprotectant, and the components were exposed to these compounds for extended periods during the injection / removal step. Further strategies were pursued where the vitrification solution was highly concentrated, while a lower concentration of cryoprotectant was used in the injection / removal step (i.e., injection / removal at VS55, but the components were vitrified at VS70). In this way, the components were exposed to a low overall concentration of cryoprotectant for an extended period, and only briefly to the full concentration of the solution during vitrification. A series of vitrification solutions with several variations in the injection / removal strategy were evaluated (see Table 2).

[0128] [Table 2]

[0129] This strategy was highly effective, yielding good survival rates immediately after reheating, and these rates persisted until the second day after reheating. The solution combination that provided the best survival rate is highlighted in Table 2 (above).

[0130] Further testing of the epidermal components showed that the vitrified components reacted similarly to fresh controls when subjected to toxicity testing with Triton-X100. The results are shown in Figure 5.

[0131] Survival rates, measured using the MTT assay, were similar for vitrified and fresh epidermal components exposed to Triton-X100 for up to 12.5 hours. Additional tests were performed to evaluate IL-1α release compared to fresh controls. Several vitrified solutions were tested (see samples marked with * in Table 2), and IL-1α release appeared to be somewhat dependent on the injection / removal combination of the vitrification solution used. Overall, the release was comparable to that of fresh components cultured for similar periods (see results in Table 3).

[0132] [Table 3]

[0133] The initial experiments were conducted using glass vials, but further experiments were performed using the deep-well plates described herein (Table 2). This allowed for the vitrification of multiple components simultaneously. In the initial experiments, 4 to 6 components were vitrified per plate. Subsequently, further experiments (repeated several times) were conducted, and it became possible to vitrify 24 components simultaneously, yielding good results demonstrating consistent viability across the entire plate.

[0134] Furthermore, several other components were also vitrified in glass vials and deep-well plates, including not only the epidermis but also EpiAirway, EpiOcular, and EpiCorneal, among other components. All of these components responded well to vitrification and remained viable for several days after reheating. The results are shown in Figure 6.

[0135] While the components were successfully vitrified in glass vials, it was observed that vitrification using the deep-well plates of this disclosure resulted in more stable survival rates. However, when using deep-well plates, timing is crucial to prevent the components from remaining in the highest-intensity vitrification solution for too long.

[0136] Additional experiments were conducted using deep-well plates to confirm the ability to vitrify and preserve several components at temperatures above -135°C for up to 7 months. The results are shown in Figure 7 below (epidermis: 6 months, EpiAirway: 7 months, EpiOcular: 2 months).

[0137] Each component showed a good survival rate (over 85%) immediately after reheating, which lasted for several days (over 70%). The initial survival rate immediately after reheating using glass vials was similar, but the sustained survival rate several days after reheating was inconsistent and not good, at approximately 45%.

[0138] To vitrify multiple components simultaneously without worrying about them remaining in the vitrification solution for too long and affecting their viability, a cassette was designed to hold up to 24 components at a time, allowing the vitrification step to be performed on multiple components simultaneously. Epidermal components were vitrified in either a deep-well plate or the cassette. After reheating, metabolic activity was measured. It was observed that components vitrified using the cassette exhibited comparable viability to those vitrified in a deep-well plate. The results are shown in Figure 8.

[0139] All documents and patent referenced throughout this disclosure are incorporated in their entirety by reference. The foregoing descriptions are made herein by reference to specific means, materials, and embodiments, but are not intended to be limited to any specific disclosed herein, but rather to encompass all functionally equivalent structures, methods, and uses, such as those within the scope of the appended claims. Furthermore, although only a few exemplary embodiments have been described in detail above, those skilled in the art will readily understand that many modifications are possible in the exemplary embodiments without substantially departing from the disclosure of “Cassets for Preserving Natural and Biological Tissues.” Accordingly, all such modifications are intended to be within the scope of this disclosure as defined in the following claims. In the claims, the means-plus-function clause is intended to cover the structures described herein as performing the cited function, and is intended to cover not only structural equivalents but also equivalent structures. Thus, nails and screws may not be structurally equivalent in that nails use a cylindrical surface to fasten wooden parts, while screws use a helical surface; however, in the context of fastening wooden parts, nails and screws may be equivalent structures. Except where a claim explicitly uses the term “means for” in conjunction with the function it relates to, it is the express intention of the applicant not to invoke § 112(f) of the U.S. Patent Act to limit the claims herein.

Claims

1. A multi-sample cassette comprising a first part and a second part configured to engage with each other via a releasable locking mechanism, wherein the second part comprises one or more inserts and a plurality of placeholders, each of the plurality of placeholders configured to receive one of the one or more inserts, Each of the one or more inserts comprises at least one biotechnological component or natural tissue sample. The first and second portions include a plurality of openings configured to allow the solution to flow in and around each of the one or more inserts and the at least one biotechnological component or natural tissue sample during the cryopreservation protocol, wherein the plurality of openings included in the second portion is fewer than the plurality of openings included in the first portion, and Each of the plurality of placeholders is located on the surface of the second portion, between adjacent openings among the plurality of openings included in the second portion. There is no inner wall between adjacent placeholders among the aforementioned plurality of placeholders. The configuration of the placeholder within the cassette corresponds to the configuration of the wells in a plate having a predetermined number of wells, and the predetermined number of wells is in the range of 6 to 384. Multi-sample cassette.

2. A multi-sample cassette according to claim 1, wherein the first portion has a frame with a thickness in the range of 1 to 2 mm.

3. A multi-sample cassette according to claim 1, wherein the second portion is a multi-sample cassette having a frame with a thickness in the range of 1 to 2 mm.

4. A multi-sample cassette according to claim 1, wherein each of the plurality of openings has a diameter in the range of 4 to 6 mm.

5. A multi-sample cassette according to claim 1, wherein each of the plurality of openings is spaced apart from the nearest adjacent opening by a distance of 3 to 15 mm.

6. A multi-sample cassette according to claim 1, wherein each of the plurality of openings has a diameter in the range of 4 to 6 mm, and the diameter of each of the plurality of openings is the same in both the first and second portions.

7. A multi-sample cassette according to claim 1, wherein each of the plurality of openings has a diameter in the range of 4 to 6 mm, and the diameter of each of the plurality of openings in the first portion is different from the diameter of each of the plurality of openings in the second portion.

8. A multisample cassette according to claim 1, wherein the frame of the first portion and the frame of the second portion are opaque.

9. A multisample cassette according to claim 1, wherein the frame of the first part and the frame of the second part are transparent.

10. A multi-sample cassette according to claim 1, wherein the frame of the first part and the frame of the second part are made of polypropylene, polystyrene, or Teflon.

11. A multi-sample cassette according to claim 1, wherein the first portion and the second portion are formed from plastic.

12. A multi-sample cassette according to claim 1, wherein one or more inserts are made of polypropylene, polystyrene, or Teflon.

13. In the multi-sample cassette according to claim 1, the area inside the cassette is 89 cm². ２ ~101cm ２ A multi-sample cassette, which is within the range of a multi-sample cassette.

14. In the multi-sample cassette according to claim 1, the volume of the at least one biotechnological component or natural tissue sample is 1 cm³. ３ ~1.5cm ３ A multi-sample cassette, which is within the range of a multi-sample cassette.

15. In the multi-sample cassette according to claim 1, the total volume of the internal chamber of the multi-sample cassette is 81 cm³. ３ ~98cm ３ A multi-sample cassette, which is within the range of a multi-sample cassette.

16. A multi-sample cassette according to claim 1, wherein the at least one biotechnological component or natural tissue sample is fixed / resistible to the surface of one or more inserts.

17. A multi-sample cassette according to claim 1, wherein each of the multiple openings is circular or hexagonal in shape.

18. A multi-sample cassette according to claim 1, wherein each side wall of the second portion is provided with the plurality of openings, and each of the plurality of openings in the side wall of the second portion is square or rectangular in shape.

19. A multi-sample cassette according to claim 1, wherein the at least one biotechnological component or natural tissue sample includes immobilized living cells.

20. A multi-sample cassette according to claim 19, wherein the at least one biotechnological component or natural tissue sample is obtained from natural or artificial tissue or organ.

21. A multi-sample cassette according to claim 1, wherein the cassette is reusable.

22. A multi-sample cassette according to claim 1, wherein the cassette is disposable.

23. A multi-sample cassette according to claim 1, wherein the frame of the first part and the frame of the second part are made of polypropylene or Teflon.

24. A multi-sample cassette according to claim 1, wherein one or more inserts are made of polypropylene or Teflon.

Images